IN PRIMARY IMMUNODEFICIENCIES

APECED: Is this a model for failure of T-cell and B-cell tolerance?

Nicolas Kluger, Annamari Ranki and Kai Krohn

Journal Name: Frontiers in

ISSN: 1664-3224

Article type: Review Article

Received on: 24 Apr 2012

Accepted on: 15 Jul 2012

Provisional PDF published on: 15 Jul 2012

Frontiers website link: www.frontiersin.org

Citation: Kluger N, Ranki A and Krohn K(2012) APECED: Is this a model for failure of T-cell and B-cell tolerance?. 3:232. doi:10.3389/fimmu.2012.00232

Article URL: http://www.frontiersin.org/Journal/Abstract.aspx?s=1244& name=primary%20immunodeficiencies&ART_DOI=10.3389 /fimmu.2012.00232

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1 2 3 4 APECED: Is this a model for failure of T-cell and B-cell tolerance? 5 6 7 8 9 10 Nicolas Kluger1, Annamari Ranki1 and Kai Krohn2* 11 12 1Department of Dermatology, Allergology and Venereology, Institute of Clinical Medicine, 13 University of Helsinki, Skin and Allergy Hospital, Helsinki University Central Hospital, Helsinki, 14 Finland 2 15 Clinical Research Institute HUCH Ltd, Helsinki, Finland 16 17 18 19 20 *Corresponding author: Professor Kai Krohn, Salmentaantie 751, 36450 Salmentaka, Finland. E- 21 mail [email protected] 22 Phone number: +358-40-833 1366 23 Fax number: +358-9-4718 6500 24 25 Running title: APECED syndrome 26 27 Conflicts of interest: none declared 28 Grant support: The study was financially supported by the European Science Foundation (ESF) and 29 the Sigrid Juselius foundation (NK) 30 31 32 Word count: 6027 33 Figure :Figure: 1 34 Tables :Tables: 2 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 2

50 Abbreviations 51 AADC: aromatic L-amino acid decarboxylase 52 AD: Addison’s disease 53 AE: Autoimmune 54 APS-1: Autoimmune polyendocrinopathy syndrome type 1 55 APECED: Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy 56 CMC: chronic mucocutaneous candidiasis 57 EECs: enteroendocrine cells 58 GI: gastro-intestinal 59 HP: Hypoparathyroidism 60 IPEX: , polyendocrinopathy, enteropathy and X-linked 61 IF: Intrinsic factor 62 IL-1: Interleukin-1 63 IL-17: Interleukin-17 64 IL-22: Interleukin-22 65 mTECs: thymic medullary epithelial cells 66 NALP-5: NACHT leucine-rich-repeat 5 67 PE : promiscuous expression 68 PTH : parathormone 69 SLE: systemic lupus erythematosus 70 TH : tyrosine hydroxylase 71 TPH :tryptophan hydroxylase 72 TRIMs: tripartite motif-containing 73 TSA : tissue specific antigens 74 3

75 Summary 76 In APECED, the key abnormality is in the T-cell defect that may lead to tissue destruction chiefly 77 in endocrine organs. Besides, APECED is characterized by high-titer antibodies against a wide 78 variety of cytokines, that could partly be responsible for the clinical symptoms during APECED, 79 mainly chronic mucocutaneous candidiasis, and linked to antibodies against Th17 cells effector 80 molecules, IL-17 and IL-22. On the other hand, the same antibodies, together with antibodies 81 against type I interferons may prevent the patients from other immunological diseases, such as 82 and systemic lupus erythematous. The same effector Th17 cells, present in the 83 lymphocytic infiltrate of target organs of APECED, could be responsible for the tissue destruction. 84 Here again, the antibodies against the corresponding effector molecules, anti-IL-17 and anti-IL-22 85 could be protective. The occurrence of several effector mechanisms (CD4+ Th17 cell and CD8+ 86 CTL and the effector cytokines IL-17 and IL-22), and simultaneous existence of regulatory 87 mechanisms (CD4+ Treg and antibodies neutralizing the effect of the effector cytokines) may 88 explain the polymorphism of APECED. Almost all the patients develop the characteristic 89 manifestations of the complex, but temporal course and severity of the symptoms vary 90 considerably, even among siblings. The autoantibody profile does not correlate with the clinical 91 picture. One could speculate that a secondary homeostatic balance between the harmful effector 92 mechanisms, and the favorable regulatory mechanisms, finally define both the extent and severity 93 of the clinical condition in the AIRE defective individuals. The proposed hypothesis that in 94 APECED, in addition to strong tissue destructive mechanisms, a controlling regulatory mechanism 95 does exist, allow us to conclude that APECED could be treated, and even cured, with 96 immunological manipulation. 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 4

124 Synopsis 125 126 Autoimmune polyendocrinopathy syndrome type 1 (APS-1), also called APECED (Autoimmune 127 polyendocrinopathy-candidiasis-ectodermal dystrophy) is a recessively inherited monogenic 128 condition, caused by in both alleles of the AIRE-gene (Autoimmune regulator). The 129 clinical hallmarks of APECED are sequentially erupting failures of endocrine organs, but other 130 manifestations, such as enamel dysgenics’, keratoconjuctivitis and asplenia occur as well. The 131 phenotype varies in individual patients as to the time course and severity of the symptoms. 132 133 AIRE is normally expressed in the medullary epithelial as well as dendritic cells in the thymus and 134 is thought to be responsible for the promiscuous expression (PE) of tissue specific antigens (TSA). 135 This process in turn would lead to the destruction of potentially auto-reactive T-cells and their 136 escape from this central tolerance mechanism into the periphery and their target organs. However, 137 recent findings have shed light for alternate mechanisms of action. In thymus, it has been proposed 138 that AIRE is responsible for the proper development and differentiation of medullary epithelial 139 cells, rather than being a factor regulating the expression of TSAs. Furthermore, in 140 addition of thymus, AIRE expression can also be seen, although in very few number, in cells 141 derived from the two other germinal layers, mesoderm and ectoderm and AIRE protein can be seen 142 by immunofluorescence in some rare cells in peripheral lymphatic tissue and in the basal cells of 143 epidermis. This extra-thymic expression may be linked to the non-endocrine lesions seen in 144 APECED. 145 146 Finally, during the recent years, two new immunological mechanisms have been shown to operate 147 in APECED. The level of regulatory T-cells, characterized by the FOXP3 gene, is low in the 148 APECED patients. Furthermore, in addition to organ specific antibodies, APECED patients have 149 autoantibodies to a large series of different cytokines, the most frequently occurring being those 150 recognizing type 1 interferons and the product of Th17 cells, IL-17 and IL-22. Antibodies to the 151 last two are thought to be responsible for the chronic candidiasis, but in general, these cytokine 152 antibodies could also be protective, forming a balance mechanism to counter the effect of the 153 effector molecule. 154 155 In summary, both T-cell and B-cell products seem to be responsible for the various clinical signs 156 and symptoms in APECED. In an effort to balance these detrimental immunological mechanisms, 157 there seems to be again both T- and B-cell linked mechanisms, the regulatory T-cells and anti- 158 cytokine antibodies. 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 5

175 Introduction: 176 Autoimmune polyendocrinopathy syndrome, type 1 (APS-1) or autoimmune polyendocrinopathy– 177 candidiasis–ectodermal dystrophy syndrome (APECED; OMIM 240300) is a rare recessively 178 inherited disorder [1-4]. It is caused by mutations in the autoimmune regulator (AIRE) gene located 179 on locus 21q22.3 [5-7]. APECED displays a worldwide distribution, but specific clusters of high 180 prevalence of the disease are observed among Finns (1 : 25 000) [8] and Sardinians 1(1 : 14 500) 181 [9,10]. It is characterized by the variable association of autoimmune endocrine 182 (hypoparathyroidism, Addison’s disease, hypothyroidism, gonadal insufficiency, insulin-dependent 183 diabetes mellitus, atrophic gastritis and Biermer’s disease) and non-endocrine disorders (keratitis, 184 malabsorption, vitiligo, alopecia areata) and a specific predisposition to chronic mucocutaneous 185 candidiasis (CMC). A definite diagnosis of APECED is made upon one of the following criteria: i) 186 the presence of at least two of three major clinical features: CMC, hypoparathyroidism (HP) and 187 Addison's disease (AD), or ii) one disease component if a sibling has already a definite diagnosis or 188 iii) disease-causing mutations in both alleles of the AIRE gene. However, APECED being highly 189 variable in its presentation, the classical triad may be complete only after years of evolution and 190 diagnose may be therefore missed. Besides, APECED may appear during adolescence or in the 191 young adult [4]. Therefore, criteria for a probable APECED have been defined as follows: i) 192 presence of one of CMC, HP, AD (before 30 years of age) and at least one of the minor components 193 chronic diarrhea, keratitis, periodic rash with fever, severe constipation, autoimmune hepatitis, 194 vitiligo, alopecia, enamel hypoplasia, ii) any component and anti-interferon antibodies or iii) any 195 component and antibodies against NALP5 , AADC, TPH or TH [4]. 196 197 From circulating auto-immune antibodies to AIRE, FOXP3, APECED and IPEX 198 199 Our knowledge of the nature of the condition now called APS-1 or APECED has increased 200 simultaneously with the general development of immunology and . Since the 201 condition was clearly defined in the end of 1950´s and early 1960´s, the characteristic clinical 202 picture, the immunological abnormalities and the relationship to other autoimmune endocrine 203 diseases were defined in late 1960´s and early 1970´s. Furthermore, the genetics of APECED, and 204 the fact that the syndrome was caused by a recessive gene defect - as opposed to the HLA linked 205 genetics seen in the other solitarily occurring endocrine diseases - were characterized in the 1980’s 206 and the target antigens in the organs affected by APECED were molecularly defined in 1990´s. A 207 landmark stage in the study of APECED was reached in 1997, when the long sought APECED gene 208 was cloned by two independent groups [5,6]. Finally, a new phase in APECED research occurred 209 during the first decennium of 2000, when the autoantibodies towards soluble mediators if immune 210 response were characterized [11,12]. 211 212 The notion that several diseases affecting endocrine organs and earlier defined as idiopathic, were 213 in fact caused by an autoimmune response towards self antigens, became apparent when novel 214 immunological methods became available in 1950´s and early 1960´s [13]. The association of the 215 three conditions, candidiasis, HP and AD, that were later judged to be the hallmarks of APECED 216 was clearly stated by the groups of Blizzard and Maclaren [13-15]. These groups also defined two 217 clearly distinct syndromes with several associated autoimmune diseases: Autoimmune 218 polyglandular syndrome type 1 (PGS-1) and polyglandular syndrome type 2 (PGS-2). The 219 nomenclature was later changed to autoimmune polyendocrinopathy type 1 and 2 (APS-1 and APS- 220 2) and the former further to APECED [1-3,8]. 221 222 Pioneering studies in this field were made especially with the use of immunohistochemistry, 223 demonstrating antibodies reacting with gastric parietal cells in chronic gastritis [16,17] and intrinsic 224 factor in pernicious anemia [18,19], with thyroid epithelial cells in various forms of thyroid diseases 6

225 [20-22], with the beta-cells of Langerhans islands in diabetes mellitus [23,24] and adrenal cortical 226 cells in Addisons disease [25]. 227 228 Blizzard´s group noticed that the two polyglandular syndromes, APS-1 and APS-2, differed in their 229 HLA haplotypes [15]. Further studies on the HLA haplotypes revealed that the genetic basis of 230 APS-2, but also of the other isolated forms of endocrine autoimmune diseases found in APS-1, were 231 in the HLA haplotype of the patients. In contrast, APS-1 was shown not to be linked to HLA, and 232 studies with large patient material, collected by Jaakko Perheentupa´s group in Finland, clearly 233 stated that APS-1 was linked to a recessively inherited gene defect [1,3,8]. The autoimmune 234 endocrinopathies could thus be grouped on the bass of their genetic background in two distinct 235 categories: those linked to HLA variation and the one, APS-1 caused by a single mutated gene 236 (Table 1). At that stage, however, the responsible gene, the APECED gene, was not yet identified. 237 Once identified, the APECED gene was renamed as AIRE (Autoimmune regulator) in 1997 [5,6]. 238 239 Another immunopathy, termed originally as (AIE) and later identified as 240 Immune dysregulation, polyendocrinopathy, enteropathy and X-linked (IPEX), was described in the 241 1980´s and 1990´s (26). This disorder was later shown to be caused by a defect in a single gene, 242 FOXP3 (27). IPEX and APECED are two examples of immune deficiency diseases disclosing both 243 disturbed tolerance and autoimmune phenomena [28]. Traditionally, reviews tend to associate both 244 IPEX and APECED because of common features. However, both clinical manifestations and 245 predisposition to infections are rather different when comparing both diseases [28]. 246 247 AIRE gene, mutations and mechanism of action 248 249 AIRE is expressed in thymus, lymph nodes and fetal liver, and encodes a protein with two putative 250 zinc fingers and other motifs suggestive of a transcriptional regulator (5,6) The AIRE gene, 251 approximately 13 kb in length, contains 14 exons that encode a polypeptide of 545 amino acids. 252 The AIRE protein functions as a [29,30]. AIRE is expressed in the thymic 253 medullary epithelial cells (mTECs) and in cells of the monocyte/dendritic cell lineage [31]. mTECs 254 through the expression of MHC class II express a wide array of tissue-restricted antigens (TRAs) 255 derived from different organs in the body. TRAs include self-proteins with patterns of expression 256 restricted to a single or small handful of organs. Thymic expression of TRA serves as an important 257 source of self-antigens to allow the negative selection of autoreactive T cells. Collectively, mTEC 258 and thymic monocyte/dendritic cells play a crucial role in establishing self-tolerance by eliminating 259 autoreactive T cells (negative selection) and/or by producing immunoregulatory FOXP3+ T cells, 260 which prevent CD4+ - mediated organ-specific autoimmune diseases. Collectively, several 261 studies in mouse and man have shown that AIRE regulates thymic expression of several genes of 262 ectopic peripheral proteins including many TRAs. Thus, AIRE dysfunction leads to a decrease in 263 the expression of TRAs in the thymus, and consequently, autoreactive T-cell clones escape into the 264 periphery [28-30,32] 265 266 The most common AIRE , the “Finnish mutation”, R257X, affects 82% of Finnish 267 APECED alleles [5,6]. Interestingly, this mutation occurs also in 70 % of the Russian APECED 268 patients studied [33]. The same mutation, R257X was also detected in Swiss patients on a different 269 haplotype with closely linked polymorphic markers [5] and in northern Italian APECED patients . 270 Nonsense mutation R139X was found as the predominant haplotype among Sardinian patients 271 (18/20 independent alleles) [9]. Other hotspots have been identified such as the Y85C missense 272 mutation in an isolated Iranian Jewish community [34,35]. A 13-bp deletion in exon 8 (1085– 273 1097(del)) is ubiquitous and can be found in Norwegians, but also Anglo-Saxons descendant [34] 7

274 and south Americans [28]. Today, over 60 different mutations have been described throughout the 275 coding region of AIRE [36]. 276 277 278 279 Clinical picture of APECED. 280 281 The clinical picture of APECED is characterized by sequentially occurring diseases, with great 282 variation among the patients as to the severity and time course of the various conditions. In most 283 cases, the affected individual starts suffering from CMC in early infancy or childhood. In most 284 cases, the next organs to be affected, are the parathyroid glands, followed by AD and at puberty, 285 hypogonadism mainly in female teens or young adults. Additional clinical features are less 286 common, and include diabetes type I, hypothyroidism, atrophic gastritis with or without pernicious 287 anemia (Biermer’s disease), cutaneous manifestations (alopecia areata, vitiligo, transient skin rash 288 during fever episodes, non-infectious nail dysplasia), ocular symptoms (keratoconjunctivitis, dry 289 eye, iridocyclitis, cataract, retinal detachment and optic atrophy)[37], enamel dysplasia, 290 hyposplenism/asplenia (implying vaccination against Streptococcus pneumonia, Haemophilus 291 influenzae and Hepatitis B as well as antibiotic prophylaxis), autoimmune hepatitis, tubulo- 292 interstitial nephritis or organized pneumonitis. Involvement of the gastro-intestinal (GI) tract may 293 be responsible for chronic diarrhea, constipation, and malabsorption leading sometimes to 294 malnutrition. GI involvement is difficult to assess as it can be due to numerous various causes that 295 may be associated or follow each other during the life of the patients. 296 297 Candidiasis 298 CMC infection by Candida albicans is one of the major characteristic of APECED, usually one of 299 first symptoms and most likely the most disabling features of APECED. CMC is naturally not 300 specific forAPECED but any child with CMC should be suspected of APECED. According to the 301 Finnish experience, almost all adults with APECED display symptoms of CMC, up to 70% of the 302 patients at the age of 10, up to 94% at age of 20 and 97% at the age of 30 [1-4]. However, the 303 course and severity vary widely. Oral candidiasis affects the tongue, the buccal mucosa, the 304 gingival and the pharynx. It ranges in severity from mild form with redness, soreness, angular 305 cheilitis, pseudomembranous lesions, erosions, ulceration and pain to severe chronic inflammation 306 with dysphagia and development of hyperkeratotic plaques. In the absence of active antimycotic 307 treatment and careful follow-up, chronic oral candidiasis may lead to the development of squamous 308 cell carcinoma with potential lethality by metastatic dissemination. Candida oesophagitis has been 309 reported to affect 15 to 22% of the patients [3,12] with pain while swallowing, retrosternal pain and 310 dysphagia [4,8]. Chronic oesophagitis can lead to local stricture and exceptional oesophageal cancer 311 [38]. 312 313 Intestinal candidiasis may cause chronic diarrhea. It should be stressed that oesophageal and 314 intestinal candidiasis may occur without any active oral candidiasis. Genital candidiasis affects 315 mainly women with pruritus and vaginal whitish discharge while genital candidiasis seems less 316 frequent in males, possibly underreported due to discrete signs of balanitis. Lastly, candida may 317 affect the nails with chronic paronychia and onychomycosis [39]. Fingernails are more commonly 318 affected than toenails and the thumbs are the commonest digit affected. This can be explained as 319 infection occurs during the “thumbsucking” period. Management of candidiasis in APECED 320 patients implies an excellent oral hygiene with a careful and regular dental follow-up. Candidiasis 321 should be treated aggressively with antimicrobial therapy and regular prophylaxis should be given. 8

322 Any clinically suspicious, chronic thickening or erosion of the mucosa that does not heal should be 323 biopsied to rule out a potential underlying lesion of squamous cell carcinoma. Any difficulties in 324 swallowing or eating or retrosternal pain should prompt to perform oesophagoscopy [38]. 325 326 Hypoparathyoidism 327 HP is one of the first endocrine feature of APECED. Symptoms are related to hypocalcaemia : 328 muscle cramps, paraesthesia, clumsiness, seizures, diarrhea, The diagnosis is simply based on 329 blood calcium, phosphorus and parathormone (PTH) levels: hypocalcaemia, hyperphosphataemia, 330 inadapted normal/low PTH without any kidney failure. It is considered that APECED should be 331 systematically considered in cases of primary HP [4]. Antibodies against NALP5 (NACHT leucine- 332 rich repeat protein 5) as well as against the calcium-sensing receptor of parathyroid epithelial cell 333 have been identified in APECED patients [40-42]. Patients who are free of HP need an annual 334 monitoring of blood calcium and phosphorus levels. Management of HP relies on daily oral 335 supplementation of vitamin D derivatives and calcium. 336 337 338 Gastritis and pernicious anemia 339 340 Chronic gastritis, with or without concomitant pernicious anemia belongs to the APECED complex 341 but is found only in a fraction of cases. In non-APECED population, two types of chronic gastritis 342 occur, divided by Strickland into type A and B gastritis. Type A gastritis was known to be caused 343 by autoimmunity while the B gastritis was suspected to be the results of environmental factors. In 344 early 1980s, it was shown by Barry Marshall and Robin Warren [43] that the major environmental 345 factor was in fact a chronic infection with Helicobacterium pylori. 346 347 The type A chronic gastritis, with and without pernicious anemia that occur in non-APECED 348 individuals, is clearly linked to certain HLA risk haplotypes, in analogy to isolated Addison disease. 349 In APECED patients, the chronic gastritis differs from the above also in time of occurrence and the 350 speed of the progression. In non-APECED patients, the time needed for progression from the early 351 stage of gastritis, the superficial form to diffuse gastritis, to atrophic gastric and to full gastric 352 atrophy is a slow process, taking up several decennia. Also, the process usually starts in the adult 353 life. In contrast, an APECED-associated gastritic process is much faster and can start in the first 354 decennium of life. Thus, one of the authors of this review was able to follow such a gastric process 355 in two eight-year old girls with sequential gastric biopsies and could see how, within the time 356 period of only 2 months, the superficial process lead to complete gastric atrophy of the fundus and 357 corpus (K. Krohn, personal experience). 358 359 The target molecule for the parietal cell antibodies were shown to be the sodium-potassium channel 360 molecule of the parietal cells on corpus and fundal part of the stomach [44] . In antral gastritis, the 361 antigen are the gastrin - producing cells [45]. 362 363 Pernicious anaemia is the end stage of the gastric immunological destruction, caused partly by the 364 lack of intrinsic factor (IF), that in addition to the hydrochloric acid is the main product of parietal 365 cells, but also by the autoantibodies recognizing this vitamin B12-binding protein. There are two 366 types of antibodies to IF: one blocking vitamin B12 binding to IF and another type, binding to the 367 IF molecule without interfering with vitamin B12 binding [46]. Both antibody types prevent the 368 binding of IF to its receptor on the ileal mucosa and subsequent translocation of the vitamin B12 369 from ileum to circulation. 370 371 9

372 Addison´s disease 373 374 Adrenocortical failure or Addisons disease, described by Thomas Addison in the 19th century, is 375 considered one of the three hallmarks of APECED, but it occurs also as a solitary disease, or as part 376 of the APS-2 complex. Today, in western word, most cases of Addison´s disease are caused by 377 autoimmunity, but adrenal cortical destruction and subsequent cortical failure can be caused by 378 several other factors, notably by secondary tuberculosis or other chronic infections. In retrospect, 379 the cases described by Thomas Addison were most likely caused by tuberculosis. 380 381 The clinical of Addison´s disease are mostly similar in APECED and in 382 solitary Addison´s disease as well as in APS-2 complex. These include decreased levels of gluco- 383 and mineralocorticoids and elevated ACTH concentrations. The most severe consequence of 384 Addison´s disease is the life-threatening Addisonian crisis, characterized by general fatigue, 385 dizziness, diarrhea and death, if the patients is not quickly substituted with , mainly 386 hydrocortisol. 387 388 Autoantibodies to adrenal cortex are the characteristic immunological feature in Addison´s disease, 389 be it part of APECED or APS-2 or the solitary form. These antibodies can be easily demonstrated 390 by immunofluorescence. However, in APECED, but not in the other forms of Addison´s disease, 391 the autoantibodies are precipitating, and this phenomenon can be demonstrated by Ouchterlony´s 392 immunodiffusion [47-49]. In immunodiffusion with APECED serum against adrenal homogenate 393 three precipitating lines were observed, and one of these were shown to represent a mitochondrial 394 antigen while the two others were microsomal. 395 396 The nature of the adrenal cortical auto-antigens were revealed in early 1990´s and shown to be the 397 three main stereogenic enzymes, P450c17, P450c21 and P450scc [47, 50-52]. These three enzymes 398 were also shown to be the ones that could be precipitated by immunodiffusion. The autoantibodies 399 against these three steroigenic enzymes clearly distinguish the three clinical conditions with adrenal 400 failure: antibodies to all three can be found only in APECED, while in solitary Addison´s disease 401 and in APS-1, only antibodies to P450c21 are seen. Furthermore, in non-autoimmune Addison´s 402 disease, caused by tumors or chronic infections, such antibodies do not occur. 403 404 Gonadal functions 405 406 Autoimmune oophoritis is responsible for an ovarian insufficiency that may be dramatic for female 407 patients as insufficiency starting in teenagers and young adults. Patients may have either a primary 408 amenorrhoea with no or arrested puberty. Other patients develop premature menopause. The 409 diagnosis is confirmed by sexual hormones status ; elevated plasma levels of follicle stimulating 410 hormone (FSH) and luteinizing hormone (LH) and low oestrogen levels. Autoantibodies against 411 side-chain cleavage enzyme have been related to ovarian insufficiency [53] and also steroidogenic 412 enzymes antibodies against cytochrome p450 21-hydroxylase (CYP21A2), cytochrome p450 17a- 413 hydroxylase (CYP17) and cytochrome p450 side-chain cleavage enzyme (CYP11A1). 414 415 In female patients, hormonal substitution by oestrogen needs to be initiated during puberty. It is 416 strongly advised not to delay pregnancy. In case of hypogonadism, embryo donation has been tried 417 with success. 418 419 In males, testicular failure is less common and occurs later. The prevalence of hypogonadism in 420 males is three times lower (8-28%) than in females (35–70%) [3]. It leads to clinical hypogonadism 421 or isolated azoospermia [4]. It has been hypothesized that the blood-testis barrier protects the 10

422 Leydig cells from an autoimmune attack. However, the physiopathogenic link between circulating 423 autoantibodies and hypogonadism is far from being clear. The two steroidogenic enzymes, p450scc 424 and p450c17 are the main antigens in gonadal failure linked to APECED, but other potential 425 antigenic targets have been identified such as testis-expressed protein TSGA10 [54]. However, 426 despite autoantibodies directed against TSGA10 in 7% of the APECED patients, no correlation 427 could be found with gonadal failure [54]. One should not forget that the origin of gonadal 428 dysfunction may be related to an authentic specific autoimmune attack but also be related to other 429 hormonal dysfunction such as AD, pituitary insufficiency, dysthyroidism or diabetes for instance. 430 Besides, Schaller et al suggested that lack of AIRE might affect fertility by disrupting scheduled 431 apoptosis of testicular germ cells [55]. In this respect, the recent hypothesis presented by 432 Matsumoto that the function of AIRE in thymus would not be in the regulation of transcription but 433 rather in the development and differentiation of the medullary epithelial cells is of primary interest 434 [56] 435

436 437 Other endocrine disorders 438 Various other endocrine disorders have been described such diabetes type 1 mellitus, 439 hypothyroidism and pituitary failure, the latter diagnosed by a growth hormone deficiency. The 440 diagnosis and management of these conditions does not differ from the standard guidelines for each 441 disorder separately [1,2,4]. 442 443 Other non-endocrine disorders 444 Enamel hypoplasia affect mainly permanent teeth [3], but also deciduous teeth [57]. Pavlic and 445 Waltimo-Siren recently suggested that an inadequate process of enamel formation might affect all 446 ameloblasts in phase [57]. Ameloblasts have an epithelial origin with parenchymal cells of 447 endocrine origin. It is speculated that ameloblasts or secreted protein in the extracellular matrix may 448 be the target of autoantibodies leading to hypoplasia. Thereby APECED would be the first model of 449 dental hard tissue autoimmune disease [57]. 450 451 Ocular manifestations affect 25% of the patients and include mainly keratitis that can lead to 452 blindness. It is assumed that the origin of keratitis is the result of autoimmunity against corneal 453 epithelium [3,37]. However, to our knowledge no specific antibodies have been identified in 454 APECED patients. Only antibodies against OBP1 have been found in the AIRE mouse model 455 against lacrimal glands [58]. 456 457 Hyposplenism or asplenia is often diagnosed upon the development of thrombocytosis, circulating 458 Howell-Joly bodies, abdominal ultrasound imaging or in case of severe Streptococcus pneumoniae 459 infection [59]. Destruction of the spleen in APECED has been related to an autoimmune attack 460 against the spleen [1-4] although the exact mechanism remains obscure. Again, the mechanisms 461 proposed by Schaller et al, and by Mitsuro Matsumoto [55, 56] are of interest, as AIRE expression 462 has also been described in lymphoid tissue and skin. A speculative hypothesis to the evolution of 463 splenic atrophy could thus be disturbance of differentiation, due to lack of AIRE expression. 464 465 466 Various types of gastro-intestinal manifestations are common in APECED patients. These include 467 chronic diarrhea that can be related to hypoparathyroidism, severe constipation. intestinal infection 468 by candida and giardia especially, pancreatic insufficiency and autoimmune enteropathy. Several 469 Autoreactive circulating antibodies directed towards intestinal components have been described. 470 Ekwall et al identified tryptophane hydroxylase (TPH) as an intestinal autoantigen in APECED 11

471 patients [60]. TPH is expressed in serotonin-producing cells in the central nervous system and in the 472 intestine. In their series of 80 patients, they were able to relate “GI symptoms” to the presence of 473 circulating TPH antibodies and also to the total absence of enterochromaffin cells in the mucosa of 474 small bowel. These enteroendocrine cells (EECs) are scattered through the intestinal mucosa, from 475 the gastric body and antrum to the rectum. They play a key role in growth of the gut, blood flow, 476 motility, secretion of pancreatic enzymes, bile and bicarbonate rich fluid [61]. TPH antibodies were 477 found in 89% of the APECED patients with GI symptoms and in 34% of those without [60]. 478 Antibodies can precede clinical symptoms [60]. Conversely, TPH autoantibodies are absent in other 479 inflammatory or auto-immune intestinal diseases. Additionally, Sköldberg et al identified also 480 autoantibodies against histidine decarboxylase expressed by EEC – like cells in the gastric mucosa 481 [62]. It is noteworthy, that it is not a routine procedure to perform EECs staining on intestinal 482 biopsies in case of diarrhea or malabsorption, as stressed by Ohsie et al [63]. Besides, several 483 studies showed repeatedly that enteroendocrine cells were lacking in the intestinal mucosa and were 484 related to chronic diarrhea [61, 64-66]. 485 486 Tubulo-interstitial nephritis, life-threatening autoimmune bronchiolitis and other rare manifestations 487 have also been reported in APECED [1-4]. 488 489 The main identified autoantibodies are summarized in table 2. 490 491 Treatment 492 Management of APECED relies in education of the patients to know his disease, education of the 493 local physician and the knowledge that new components of the disease may develop during life. 494 Psychological support is strongly recommended as this disease impairs greatly the quality of life of 495 the patients [3]. Except candidiasis treatment that has been explained previously, treatment of 496 APECED relies mostly on hormone replacement therapy according to affected organs (thyroid, 497 parathyroid, pancreas etc.). In some rare and potentially lethal situations however, patients may 498 require treatment in association with immunosuppressive therapies. These rare 499 situations include autoimmune hepatitis, especially its fulminant form, which may be lethal and 500 therefore prompt immunosuppressive therapy is needed [67]. The same is true for interstitial 501 nephritis and bronchiolitis in association to APECED. Immunosuppressive therapies have been also 502 proposed in case of severe intestinal malabsorption with efficacy [64,65]. Very recently, rituximab, 503 a chimeric monoclonal antibody targeting B cell lymphocytes expressive CD20 has been 504 successfully used in a young patient with bronchiolitis [68]. The rationale for Rituximab use in 505 APECED is supported by the presence of B-cell infiltrates in the affected organs [69]. 506 507 508 Autoantibodies towards interferons and cytokines 509 510 At the beginning of this millennium, the antibody responses to the main target organs affected in 511 APECED, and the responsible target antigens were fairly well characterized. A new period in 512 APECED studies started along the publication by Meager et al [11], describing high-titre antibodies 513 to several type I interferons in practically all APECED patients studied. This anti-interferon 514 response was exceptionally strong, since serum titers up to 1:1 000 000, and clearly exceeding the 515 titers seen against organ-specific antigens, were found. 516 517 Furthermore, high-titre antibodies were seen against the two main mediators secreted by Th17 518 cells, interleukin-17 and interleukin -22 (IL-17 and IL-22). Responses with lower titers were 519 occasionally seen against other interleukins, too. In our own as yet unpublished observations we 520 have detected occasional high titer responses against several other interleukins and chemokines, as 12

521 well, but in contrast to the aforementioned responses, these responses are not characteristic to all 522 APECED patients but rather occur occasionally in only a few patients. 523

524 The significance of these novel findings are still unclear, but some information concerning the role 525 of IL-17/IL-22 antibodies in the chronic candida infections, characteristic for APECED, has been 526 obtained. TH17 cells secrete IL-17 and IL-22, which are cytokines with potent antifungal properties 527 [70] and the occurrence of autoantibodies against IL-17/IL-22 were reported to closely correlate to 528 the presence of candida infection [12,70]. However, recent evidence points to a new interaction 529 between AIRE and dectin-1, a pattern-recognition receptor that is important in antifungal innate 530 immunity. Pedroza et al [71] recently showed that AIRE participates in the Dectin-1 signaling 531 pathway, and thus, missing AIRE activity could contribute to fungal susceptibility through this 532 pathway. Dectin-1 is expressed on phagocytes and was recently shown to induce a noncanonical 533 caspase-8 inflammasome in response to fungal and mycobacterial infection [72]. The activation of 534 the dectin signaling pathway also leads to expression of IL-17 and 22 and defensins, however. 535 Besides, other mecanisms such Dominant-negative mutations in STAT3, gain of mutation of 536 STAT1, mutations in IL17F and IL17R may be alternate causes of CMC [70].

537 The significance of the antibody response towards interferons and other cytokines is presently also 538 unclear. One could speculate that some of these antibodies against type 1 interferons as well as 539 reacting with IL-17 and IL-22 might have a protective function. As pointed out by Waterfield and 540 Anderson [73], antibodies to type I interferons do not seem to lead to increased susceptibility to 541 viral infections. This resistance might be due to redundancy and it has to be seen whether this anti- 542 interferon response is directed only towards certain members of the interferon family. While Th17 543 cell response and the release of soluble IL-17 ad IL-22 are evidently necessary for the defense 544 against mucocutaneous candida infection, the same cytokines have a role in the development of 545 psoriasis. Similarly, interferons are known to be involved in the pathogenesis of several conditions, 546 and one such chronic ailment is the autoimmune diseases belonging to the systemic lupus 547 erythematosus (SLE) complex. Anti-interferon alpha antibodies are currently being tested as a 548 therapeutic mean against SLE [74]. In order to be able to find out if the antibodies against 549 interferons and other cytokines could have a protective role in APECED, large APECED patient 550 cohorts have to be studied in order to find out whether e.g. psoriasis and SLE are significantly less 551 common in APECED patients than in the general population. 552 553 The reason for the antibody response towards soluble immune mediators is still unclear, and we do 554 not yet know what exactly elicits them and thus, only speculative scenarios can be presented. It is 555 conceivable to hypothesize, however, that the tissue destruction preceding the failure of the 556 endocrine organs may have a role. Tissue destruction, be it caused by trauma, viral infection or 557 autoimmune attack, would probably lead not only to the release of potential tissue-specific 558 autoantigens and thus, to autoantibody formation against these proteins, but could also lead to an 559 inflammatory response and production of several mediators of inflammation. One key group of 560 molecules in this respect is the acute phase proteins, notably those belonging to the IL-1 group. 561 562 It is generally believed that the destruction of the endocrine organs in APECED is caused by the 563 autoreactive CD8+ cytotoxic T-cells, although definitive evidence for this mechanism is still 564 lacking [2,28]. This hypothesis is reinforced by the examination of microscopic examinations of 565 samples, sometimes obtained post-mortem. Indeed, parathyroid, adrenal glands or ovaries 566 pathology disclosed also atrophy and lymphocytic infiltration that suggest lymphocytic aggression 567 of the organs leading to atrophy and dysfunction [2]. This is also stressed, indirectly, by the analysis 13

568 of the AIRE deficient mouse model, who develop also a lymphocytic infiltration in some inner 569 organs along with atrophy [75]. 570 However, cell destruction caused by an immune response against the endocrine organ would in fact 571 lead to a similar situation that is thought to happen in viral infections. In fact, several autoimmune 572 diseases, such as diabetes type 1 or chronic autoimmune liver diseases are thought to be a 573 consequence of preceeding viral infection: enterovirus infection in the case of diabetes type 1 and 574 hepatitis B in the case of chronic active hepatitis. In viral infections, a specific group of intracellular 575 regulatory molecules, TRIMs (tripartite motif-containing proteins), have been shown to have a key 576 role in eliciting an autoimmune or auto inflammatory consequence [76]. 577 578 The TRIM protein family is a form of RING domain containing E3 ligases and they exert a variety 579 of biological functions, related to immunity and inflammation [76]. Specifically, of the more than 580 20 different TRIM proteins, some seem to up-regulate the expression of type I interferons and 581 proinflammatory cytokines, notable interleukin-1beta (IL-1beta). Furthermore, the same mediators 582 of immune response and inflammation are in some cases known to up-regulate the expression of 583 TRIMs. Thus, a vicious circle can theoretically occur and this in turn could lead to autoimmunity. 584 So far, overexpression of TRIMs, or an autoimmune response towards them, has been shown to be 585 linked to autoimmune and autoinflammatory processes in Sjögren’s syndrome or rheumatoid 586 arthritis [76]. 587 588 Presently, we have no information how the occurrence of autoantibodies towards the interferons 589 and other mediators of immune response might affect the aforementioned vicious circle, but it is 590 conceivable to speculate that such an antibody response could have an balancing effect. One could 591 thus form a hypothesis, that in APECED, the primary defect outside thymus, where the autoreactive 592 T-cells are not destroyed, would be the cell destruction of the endocrine organs by cell mediated 593 immune response, followed by release of cellular components taken up by professional antigen 594 presenting cells and further stimulating the activation of CD4+ Th-cells and finally resulting in an 595 autoantibody response to these organ -specific antigens. However, simultaneous overexpression of 596 TRIMs and subsequent up-regulation of a variety of soluble mediators of immune response and 597 inflammation, such as interferons and members of the IL-1 family would lead to autoantibody 598 formation also against these cytokines. Lastly, one reason for the break of tolerance to immune 599 mediators, and subsequent production of autoantibodies could be related the fact that AIRE 600 expression seems to occur, in addition to thymic epithelial cells also outside thymus, notably 601 in dendritic cells, that normally express also such mediators [77] 602 The consequences of such cytokine-directed antibody response are still an open question. In case of 603 the Th17 type interleukins (IL-17 and IL-22) there is convincing evidence that such antibodies are 604 linked to the CMC. However, at least in some cases, the antibodies may have a balancing, down 605 regulating effect on the expression of the corresponding biologically active molecules but also, by 606 regulating the immune response to target organs. Thus, it is possible to presume, that especially the 607 antibodies to type 1 interferons might have a protective effect, as some chronic immune diseases, 608 such as psoriasis and SLE, are rare or non-existing among APECED patients. 609 610 Cell-mediated immune responses 611 612 Although it is now a generally accepted view that the consequence of the AIRE defect in APECED 613 will lead to the escape of the potentially autoreactive T-cells, there is in fact rather little direct 614 evidence to show that the tissue destruction in the endocrine organs affected in APECED is caused 615 by cytotoxic CD8+ T-cells. Furthermore, most studies describing the phenotype of the lymphocytes 616 infiltrating affected organs is not from APECED patients directly, but from patients suffering of 617 solitary lesions that are similar to the ones seen in APECED, sich as solitary Addison´s disease or 14

618 diabetes. However, the solitary endocrine diseases, such as isolated thyroid disease or Addison´s 619 disease are remarkably similar in their clinical picture as well as immunological findings as those of 620 APECED. Thus, in solitary Addison´s disease and in APECED with adrenocortical failure, 621 autoantibodies recognize the p450c21 steroidogeic enzyme. Interestingly, in this disease complex 622 CD8 positive T-cells that reach against specific T-cell epitopes in p450c21 has been demonstrated 623 [78,79] Likewise, in thyroid diseases, thyroglobulin and thyroid peroxidase are recognized by the 624 autoantibodies, irrespective if the condition is occurring alone, in association of APS2 or as part of 625 the APECED complex. The similar synergism in terms of the nature of autoantigens occurs in 626 chronic immunological liver diseases, too. 627 628 629 630 In chronic aggressive hepatitis the lymphocytic infiltrating cell population has been shown to be of 631 the CD8 positive lineage [80]. In a murine model of Graves´ disease, the CD8+ cell population 632 contains also the recently identified CD8+CD122+ T cells that are functionally similar to the CD4+ 633 CD25+ regulatory T-cells [81]. Furthermore, studies in thyroid and other affected organs show that 634 one of the main cell population in the lymphocytic infiltrate are in fact the CD4+ Th17 cells that 635 secrete as effector molecules the cytokines interleukin 17 and 22 (IL-17 and IL-22). In experimental 636 autoimmune diseases, the balance between the Th17 effector cells and the two regulatory T-cells, 637 CD8+CD122+ and CD4+CD25+, seems to regulate both the occurrence and severity of tissue 638 destruction and functional failure. 639 640 There could thus be two distinct mechanism operating in the pathogenesis of autoimmunity in the 641 endocrinopathies: one mediated by soluble effector molecules, such as IL-17 and IL-22 as well as 642 type interferons, and an other one mediated by effector T-cells, which are either of the CD8+ CTL 643 cell or of the Th17 effector cell lineage. To counteract these, again two distinct biological processes 644 would occur: the production of autoantibodies and secondly, the emergency of the regulatory T- 645 cells. As to the response, it is to note that one key immunological failure in 646 APECED, is the dysregulation of the Treg cell maturation [81-86]. 647 648 In normal thymus, Treg maturation follows a preprogrammed scheme, and the immature 649 CD8+CD4+FOXP3+ seems to be prone to apoptosis, whereas the more mature form CD4+CD8- 650 FOXP3+ cells form the active Treg population [87]. According to Endharti et al [88] the 651 CD8+CXCR3+ Tregs in humans are functionally similar to murine CD8+CD122+ Tregs. 652 Furthermore, in APECED patients the recent thymic emigrant (RTE) pool of Treg cells shift to the 653 activated pool and the RTE reservoir is depleted. Most importantly, in APECED patients these cells 654 express less FOXP3 than in the healthy controls [86]. Thus, in APECED the newly formed Treg 655 cells have a developmental defect and their function is therefore impaired. Data concerning the 656 CD8+ regulatory T cells in APECED patients is missing, however. 657 658 659 The finding that the regulatory T cell population in APECED is functionally defective and that the 660 expression of the key molecule for Treg function, the FOXP3 is impaired, is consistent with clinical 661 findings in IPEX syndrome, caused by a defect in the function of the FOXP3 gene. However, it 662 should be noted that the effect of FOXP3 mutations in Treg population also in the IPEX patients is 663 highly variable. Also, in contrast to APECED there seems to be a genotype – phenotype correlation 664 in IPEX, as different mutations are associated in variable clinical picture, that show differences in 665 severity as well in the types of clinical components that are present [89,90] A consistent finding in 666 IPEX is however the inability of the CD4+CD25high Tregs to suppress the function of autologous 15

667 effector T cells [91]. There are thus several differences in the clinical picture of APECED and 668 IPEX, but both conditions show clear immune destruction of at least some endocrine organs. Both 669 conditions also share some similarities in the gastrointestinal symptoms. 670 671 The proposed hypothesis that in APECED both tissue destructive mechanisms and controlling 672 regulatory mechanisms exist raises a question whether APECED could be treated or even cured by 673 immunological manipulations. To find an answer for this question is one of the further challenges 674 for APECED research 675 676 677 678 679 Acknowledgements. We thank professors Jaakko Perheentupa, Adrian Hayday and Pärt Peterson, 680 as well as doctors Kai Kisand, Annalisa Macagno and Edward Stuart for stimulating discussions. 681 682 683 684 685 686 687 688 689 690 691 692 REFERENCES 693 694 1. Perheentupa, J. (2002). APS-I/APECED: the clinical disease and therapy. Endocrinol Metab 695 Clin North Am. 31, 295-320, vi. 696 2. Betterle, C., and Zanchetta, R. (2003). Update on autoimmune polyendocrine syndromes (APS). 697 Acta Biomed. 74, 9-33.

698 3. Perheentupa, J. (2006). Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. J 699 Clin Endocrinol Metab 91, 2843-2850. 700 4. Husebye, E.S., Perheentupa, J., Rautemaa, R., and Kämpe O. (2009). Clinical manifestations 701 and management of patients with autoimmune polyendocrine syndrome type I. J Intern 702 Med.265, 514-529. 703 5. Nagamine, K., Peterson, P., Scott, H.S., Kudoh, J., Minoshima, S., Heino, M., Krohn, K.J., 704 Lalioti, M.D., Mullis, P.E., Antonarakis, S.E., Kawasaki, K., Asakawa, S., Ito, F., and Shimizu 705 N. (1997). Positional cloning of the APECED gene. Nat Genet 17, 393–398. 16

706 6. The Finnish–German APECED Consortium. (1997). An autoimmune disease, APECED, caused 707 by mutations in a novel gene featuring two PHD-type zinc-finger domains. Nat Genet 17, 399– 708 403. 709 7. Bjorses, P., Alltonen, J., Vikman, A., Perheentupa, J., Ben-Zion, G., Chiumello, G., Dahl, N., 710 Heidemen, P., Hoorweg-Nijman, J.J., Mathivon, L., Mullis, P.E., Pohl, M., Ritzen, M., Romeo, 711 G., Shapiro, M.S., Smith, C.S., Solyom, J., Zlotogora, J., and Peltonen, L. (1996). Genetic 712 homogeneity of autoimmune polyglandular disease type I. Am J Hum Genet 59, 879–886 713 8. Ahonen, P., Myllarniemi, S., Sipila, I., Perheentupa, J. (1990). Clinical variation of autoimmune 714 polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) in a series of 68 patients. N 715 Engl J Med 322, 1829–1836. 716 9. Rosatelli, M.C., Meloni, A., Meloni, A., Devoto, M., Cao, A., Scott, H.S., Peterson, P., Heino, 717 M., Krohn, K.J., Nagamine, K., Kudoh, J., Shimizu, N., and Antonarakis, S.E. (1998). A 718 common mutation in Sardinian autoimmune polyendocrinopathy-candidiasis-ectodermal 719 dystrophy patients. Hum Genet. 103, 428-434. 720 10. Meloni, A., Willcox, N., Meager, A., Atzeni, M., Wolff, A.S., Husebye, E.S., Furcas, M., 721 Rosatelli, M.C., Cao, A., and Congia, M. (2012). Autoimmune polyendocrine syndrome type 1: 722 an extensive longitudinal study in sardinian patients. J Clin Endocrinol Metab. 97, 1114-1124. 723 11. Meager, A., Visvalingam, K., Peterson, P., Möll, K., Murumägi, A., Krohn, K., Eskelin, P., 724 Perheentupa, J., Husebye, E., Kadota, Y., and Willcox, N. (2006). Anti-interferon 725 autoantibodies in autoimmune polyendocrinopathy syndrome type 1. PLoS Med. 3, e289 726 12. Kisand, K., Lilic, D., Casanova, J.L., Peterson, P., Meager, A., and Willcox, N. (2011). 727 Mucocutaneous candidiasis and autoimmunity against cytokines in APECED and thymoma 728 patients: clinical and pathogenetic implications. Eur J Immunol. 41, 1517-1527. 729 13. Blizzard, R.M., Tomasi, T.B., and Christy, N.P. (1963). Autoantibodies against thyroid and 730 adrenal tissue in a patient with mutiple, primary endocrine deficiencies. J Clin Endocrinol 731 Metab. 23, 1179-1180. 732 14. Brun J.M. (1978). Auto-immune juvenile polyendocrinopathy (author's transl). Ann Endocrinol 733 (Paris). 39, 463-481. 734 15. Neufeld, M., Maclaren, N.K., and Blizzard, R.M. (1981). Two types of autoimmune Addison's 735 disease associated with different polyglandular autoimmune (PGA) syndromes. Medicine 736 (Baltimore).60, 355-362. 737 16. Walder, A.I., Lunseth, J.B., and Peter, E.T. (1963). Antibodies to the gastric parietal cell. Surg 738 Forum. 14, 327-328. 17

739 17. Irvine, W.J., Davies, S.H., Teitelbaum, S., Delamore, I.W., and Williams, A.W. (1965). The 740 clinical and pathological significance of gastric parietal cell antibody. Ann N Y Acad Sci. 124, 741 657-691. 742 18. Schwartz, M. (1961). Antibodies to intrinsic factor. Acta Allergol. 16, 263-266. 743 19. Jeffries, G.H., Hoskins, D.W., and Sleisenger, M.H. (1962). Antibody to intrinsic factor in 744 serum from patients with pernicious anemia. J Clin Invest.41, 1106-1115. 745 20. Witebsky, E., Rose, N.R., Paine, J.R., and Egan, R.W. (1957). Thyroid-specific autoantibodies. 746 Ann N Y Acad Sci. 69, 669-677. 747 21. Irvine, W.J., MacGregor, A.G., and Stuart, A.E. (1962). The prognostic significance of thyroid 748 antibodies in the management of thyrotoxicosis. Lancet. 2, 843-847. 749 22. Doniach, D., and Roitt, I.M. (1964). An evaluation of gastric and thyroid auto-immunity in 750 relatation to hematologic auto-immunity. Semin Hematol. 1, 313-343. 751 23. Kaldany, A. (1979). Autoantibodies to islet cells in diabetes mellitus. Diabetes. 28, 102-105. 752 24. Bottazzo, G.F., Dean, B.M., Gorsuch, A.N., Cudworth, A.G., and Doniach, D. (1980). 753 Complement-fixing islet-cell antibodies in type-I diabetes: possible monitors of active beta-cell 754 damage. Lancet. 1, 668-672. 755 25. Blizzard, R.M., Chandler, R.W., Kyle, M.A., and Hung, W. (1962). Adrenal antibodies in 756 Addison's disease. Lancet.2, 901-903. 757 26. Powell, B.R., Buist, N.R., and Stenzel, P. (1982). A X-linked syndrome of diarrhea, 758 polyendocrinopathy, and fatal infection in infancy. J Pediatr 100, 731–737. 759 27. Bennett, C.L., Yoshioka, R., Kiyosawa, H., Barker, D.F., Fain, P.R., Shigeoka, A.O., and 760 Chance, P.F. (2000). X-Linked syndrome of polyendocrinopathy, immune dysfunction, and 761 diarrhea maps to Xp11.23–Xq13.3. Am J Hum Genet 66, 461–468. 762 28. Moraes-Vasconcelos, D., Costa-Carvalho, B.T., Torgerson, T.R., and Ochs, H.D. (2008). 763 Primary immune deficiency disorders presenting as autoimmune diseases: IPEX and APECED. 764 J Clin Immunol. 28, S11-S19. 765 29. Fierabracci, A. (2011) Recent insights into the role and molecular mechanisms of the 766 autoimmune regulator (AIRE) gene in autoimmunity Autoimmunity Reviews 10, 137–143. 767 30. Gardner, J.M., Fletcher, A.L., Anderson, M.S., and Turley, S.J. (2009). AIRE in the thymus and 768 beyond. Curr Opin Immunol. 21, 582–589 769 31. Kogawa, K., Nagafuchi, S., Katsuta, H., Kudoh, J., Tamiya, S., Sakai, Y.,Shimizu, N., and 770 Harada, M. (2002). Expression of AIRE gene in peripheral monocyte/dendritic cell lineage. 771 Immunol Lett 2002 80, 195–198. 18

772 32. Derbinski, J., Gäbler, J., Brors, B., Tierling, S., Jonnakuty, S., Hergenhahn, M., Peltonen, L., 773 Walter, J., and Kyewski, B. (2005) Promiscuous gene expression in thymic epithelial cells is 774 regulated at multiple levels. J Exp Med. 202, 33-45. 775 33. Orlova, E.M., Bukina, A.M., Kuznetsova, E.S., Kareva, M.A., Zakharova, E.U., Peterkova, 776 V.A., and Dedov, I.I. (2010) Autoimmune polyglandular syndrome type 1 in Russian patients: 777 clinical variants and autoimmune regulator mutations. Horm Res Paediatr 73:449-457. 778 34. Zlotogora, J., Shapiro, M.S. (1992). Polyglandular autoimmune syndrome type I among Iranian 779 Jews. J Med Genet 29, 824–826. 780 35. Björses, P., Halonen, M., Palvimo, J.J., Kolmer, M., Aaltonen, J., Ellonen, P., Perheentupa, J., 781 Ulmanen, I., and Peltonen, L. (2000). Mutations in the AIRE gene: effects on subcellular 782 location and transactivation function of the autoimmune polyendocrinopathy-candidiasis- 783 ectodermal dystrophy protein. Am J Hum Genet 66, 378–392. 784 36. Akirav, E.M., Ruddle, N.H., and Herold, K.C. (2011). The role of AIRE in human autoimmune 785 disease. Nat Rev Endocrinol 7, 25-33. 786 37. Merenmies, L., and Tarkkanen, A. (2000). Chronic bilateral keratitis in autoimmune 787 polyendocrinopathy-candidiadis-ectodermal dystrophy (APECED). A long-term follow-up and 788 visual prognosis. Acta Ophthalmol Scand. 78, 532-535. 789 38. Rautemaa, R., Hietanen, J., Niissalo, S., Pirinen, S., and Perheentupa, J. (2007). Oral and 790 oesophageal squamous cell carcinoma--a complication or component of autoimmune 791 polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED, APS-I). Oral Oncol 43, 607- 792 613. 793 39. Collins, S.M., Dominguez, M., Ilmarinen, T., Costigan, C., and Irvine, A.D. (2006). 794 Dermatological manifestations of autoimmune polyendocrinopathy-candidiasis-ectodermal 795 dystrophy syndrome. Br J Dermatol 154, 1088-1093. 796 40. Gavalas, N.G., Kemp, E.H., Krohn, K.J., Brown, E.M., Watson, P.F., and Weetman, A.P. 797 (2007). The calcium-sensing receptor is a target of autoantibodies in patients with autoimmune 798 polyendocrine syndrome type 1. J Clin Endocrinol Metab 92, 2107-2114. 799 41. Kemp, E.H., Gavalas, N.G., Krohn, K.J., Brown, E.M., Watson, P.F., and Weetman, A.P. 800 (2009). Activating autoantibodies against the calcium-sensing receptor detected in two patients 801 with autoimmune polyendocrine syndrome type 1. J Clin Endocrinol Metab 94, 4749-4756. 802 42. Kemp, E.H., Gavalas, N.G, Akhtar, S., Krohn, K.J., Pallais, J.C., Brown, E.M., Watson, P.F., 803 and Weetman, A.P. (2010). Mapping of human autoantibody binding sites on the calcium- 804 sensing receptor. J Miner Res 25, 132-140. 19

805 43. Warren, J.R., and Marshall, B.J. (1984). Unidentified curved bacilli in the stomach of patients 806 with gastritis and peptic ulceration. Lancet 1, 1311-1315. 807 44. Karlsson, F.A., Burman, P., Lööf, L., and Mårdh, S. (1988). Major parietal cell antigen in 808 autoimmune gastritis with pernicious anemia is the acid-producing H+,K+-adenosine 809 triphosphatase of the stomach. J Clin Invest. 81, 475-479. 810 45. Uibo, R.M., and Krohn, K.J. (1984). Demonstration of gastrin cell autoantibodies in antral 811 gastritis with avidin-biotin complex antibody technique. Clin Exp Immunol 58, 341-347 812 46. Toh, B.H., van Driel, I.R., and Gleeson, P.A. (1997). Pernicious anemia. N Engl J Med 337, 813 1441-1448. 814 47. Krohn, K., Perheentupa, J., and Heinonen, E. (1974). Precipitating anti-adrenal antibodies in 815 Addison's disease. Clin Immunol Immunopathol 3, 59-68. 816 48. Heinonen, E., Krohn, K., Perheentupa, J., Aro, A., and Pelkonen, R. (1976). Association of 817 precipitating anti-adrenal anti-adrenal antibodies with moniliasis-polyendocrinopathy syndrome. 818 Ann Clin Res 8, 262-265. 819 49. Andrada, J.A., Bigazzi, P.L., Andrada, E., Milgrom, F., and Witebsky, E. (1968). Serological 820 investigations on Addison's disease. JAMA 206, 1535-1541. 821 50. Uibo, R., Aavik, E., Peterson, P., Perheentupa, J., Aranko, S., Pelkonen, R., and Krohn, K.J. 822 (1994). Autoantibodies to cytochrome P450 enzymes P450scc, P450c17, and P450c21 in 823 autoimmune polyglandular disease types I and II and in isolated Addison's disease. J Clin 824 Endocrinol Metab 78, 323-328. 825 51. Uibo, R., Perheentupa, J., Ovod, V., and Krohn, K.J. (1994). Characterization of adrenal 826 autoantigens recognized by sera from patients with autoimmune polyglandular syndrome (APS) 827 type I. J Autoimmun 7, 399-411. 828 52. Winqvist, O., Gustafsson, J., Rorsman, F., Karlsson, F.A., Kämpe, O. (1993). Two different 829 cytochrome P450 enzymes are the adrenal antigens in autoimmune polyendocrine syndrome 830 type I and Addison's disease. J Clin Invest. 92, 2377-2385. 831 53. Soderbergh, A., Myhre, A.G., Ekwall, O., Gebre-Medhin, G., Hedstrand, H., Landgren, E., 832 Miettinen, A., Eskelin, P., Halonen, M., Tuomi, T., Gustafsson, J., Husebye, E.S., 833 Perheentupa,J., Gylling, M., Manns, M.P., Rorsman, F., Kämpe, O., and Nilsson, T. (2004). 834 Prevalence and clinical associations of 10 defined autoantibodies in autoimmune polyendocrine 835 syndrome type I. J Clin Endocrinol Metab 89, 557–562. 836 54. Reimand, K,. Perheentupa, J., Link, M., Krohn, K., Peterson, P., and Uibo, R. (2008). Testis- 837 expressed protein TSGA10 an auto-antigen in autoimmune polyendocrine syndrome type I. Int 838 Immunol 20, 39-44. 20

839 55. Schaller, C.E., Wang, C.L., Beck-Engeser, G., Goss, L., Scott, H.S., Anderson, M.S., and Wabl, 840 M. (2008). Expression of Aire and the early wave of apoptosis in spermatogenesis. J Immunol 841 180, 1338–1343. 842 56. Matsumoto, M. (2011). Contrasting models for the roles of Aire in the differentiation program 843 of epithelial cells in the thymic medulla. Eur J Immunol 41, 12-17. 844 57. Pavlic, A., and Waltimo-Sirén, J. (2009). Clinical and microstructural aberrations of enamel of 845 deciduous and permanent teeth in patients with autoimmune polyendocrinopathy-candidiasis- 846 ectodermal dystrophy. Arch Oral Biol 54, 658-665. 847 58. DeVoss, J.J., LeClair, N.P., Hou, Y., Grewal, N.K., Johannes, K.P., Lu, W., Yang, T., Meagher, 848 C., Fong, L., Strauss, E.C., and Anderson, M.S. (2010) An autoimmune response to odorant 849 binding protein 1a is associated with dry eye in the Aire-deficient mouse. J Immunol 184, 4236- 850 4246. 851 59. Pollak, U., Bar-Sever Z., Hoffer, V., Marcus, N., Scheuerman, O., and Garty, B.Z. (2009) 852 Asplenia and functional hyposplenism in autoimmune polyglandular syndrome type 1. Eur J 853 Pediatr. 168, 233-235. 854 60. Ekwall, O., Hedstrand, H., Grimelius, L., Haavik, J., Perheentupa, J., Gustafsson, J., Husebye, 855 E., Kämpe, O., and Rorsman, F. (1998). Identification of tryptophan hydroxylase as an intestinal 856 autoantigen. Lancet 352, 279-283. 857 61. Posovszky, C., Lahr, G., von Schnurbein, J., Buderus, S., Findeisen, A., Schröder, C., Schütz, 858 C., Schulz, A, Debatin, K.M., Wabitsch, M., and Barth, T.F. (2012). Loss of Enteroendocrine 859 Cells in Autoimmune-Polyendocrine-Candidiasis-Ectodermal-Dystrophy (APECED) Syndrome 860 with Gastrointestinal Dysfunction. J Clin Endocrinol Metab 97, E292-E300. 861 62. Sköldberg, F., Portela-Gomes, G.M., Grimelius, L., Nilsson, G., Perheentupa, J., Betterle, C., 862 Husebye, E.S., Gustafsson, J., Rönnblom, A., Rorsman, F., and Kämpe, O. (2003). Histidine 863 decarboxylase, a pyridoxal phosphate-dependent enzyme, is an autoantigen of gastric 864 enterochromaffin-like cells. J Clin Endocrinol Metab 88, 1445-1452. 865 63. Ohsie, S., Gerney, G., Gui, D., Kahana, D., Martín, M.G., and Cortina, G. (2009). A paucity of 866 colonic enteroendocrine and/or enterochromaffin cells characterizes a subset of patients with 867 chronic unexplained diarrhea/malabsorption. Hum Pathol 40, 1006-1014. 868 64. Padeh, S,. Theodor, R., Jonas, A., and Passwell, J.H. (1997). Severe malabsorption in 869 autoimmune polyendocrinopathy-candidosis-ectodermal dystrophy syndrome successfully 870 treated with immunosuppression. Arch Dis Child 76, 532-534. 871 65. Ward, L., Paquette, J., Seidman, E., Huot, C., Alvarez, F., Crock, P., Delvin, E., Kämpe, O., and 872 Deal, C. (1999). Severe autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy in 21

873 an adolescent girl with a novel AIRE mutation: response to immunosuppressive therapy. J Clin 874 Endocrinol Metab 84, 844-852. 875 66. Oliva-Hemker, M., Berkenblit, G.V., Anhalt, G.J,, and Yardley, J.H. (2006). Pernicious anemia 876 and widespread absence of gastrointestinal endocrine cells in a patient with autoimmune 877 polyglandular syndrome type I and malabsorption. J Clin Endocrinol Metab 91, 2833-2838. 878 67. Obermayer-Straub, P., Perheentupa, J., Braun, S., Kayser, A., Barut, A., Loges, S., Harms, A., 879 Dalekos, G., Strassburg, C.P., and Manns, M.P. (2001). Hepatic autoantigens in patients with 880 autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. Gastroenterology. 121, 881 668-677. 882 68. Popler, J., Alimohammadi, M., Kämpe, O., Dalin, F., Dishop, M.K., Barker, J.M., Moriarty- 883 Kelsey, M., Soep, J.B., and Deterding, R.R. (2012). Autoimmune polyendocrine syndrome type 884 Utility of KCNRG autoantibodies as a marker of active pulmonary disease and successful 885 treatment with rituximab. Pediatr Pulmonol 47, 84-87. 886 69. Gavanescu, I., Benoist, C., and Mathis, D. (2008) B cells are required for Aire-deficient mice to 887 develop multi-organ autoinflammation: A therapeutic approach for APECED patients. Proc Natl 888 Acad Sci U S A 105, 13009-13014. 889 70. Engelhardt, K.R., and Grimbacher, B.(2012)Mendelian traits causing susceptibility to 890 mucocutaneous fungal infections in human subjects. J Allergy Clin Immunol.129, 294-305. 891 71. Pedroza, L.A., Kumar, V., Sanborn, K.B., Mace, E.M., Niinikoski, H., Nadeau, K., Vasconcelos 892 Dde, M., Perez, E., Jyonouchi, S., Jyonouchi, H., Banerjee, P.P., Ruuskanen, O., Condino-Neto, 893 A., and Orange, J.S. (2012). Autoimmune regulator (AIRE) contributes to Dectin-1-induced 894 TNF-α production and complexes with caspase recruitment domain-containing protein 9 895 (CARD9), spleen tyrosine kinase (Syk), and Dectin-1. J Allergy Clin Immunol 129, 464-472, 896 472.e1-3. 897 72. Gringhuis, S.I., Kaptein, T.M., Wevers, B.A., Theelen, B, van der Vlist, M., Boekhout, T., and 898 Geijtenbeek, T.B. (2012). Dectin-1 is an extracellular pathogen sensor for the induction and 899 processing of IL-1β via a noncanonical caspase-8 inflammasome. Nat Immunol 13, 246-254. 900 73. Waterfield, M, and Anderson, MS. (2011) Autoimmunity's collateral damage: 901 Immunodeficiency hints at autoreactivity to cytokines. Nat Med. 17:1054-1055. 902 74. Merrill, J.T., Wallace, D.J., Petri, M., Kirou, K.A., Yao, Y., White, W.I., Robbie, G., Levin, R., 903 Berney, S.M., Chindalore, V., Olsen, N., Richman, L,. Le, C., Jallal, B., and White, B.; Lupus 904 Interferon Skin Activity (LISA) Study Investigators. (2011). Safety profile and clinical activity 905 of sifalimumab, a fully human anti-interferon α monoclonal antibody, in systemic lupus 22

906 erythematosus: a phase I, multicentre, double-blind randomised study. Ann Rheum Dis 70, 1905- 907 1913. 908 75. Ramsey, C., Winqvist, O., Puhakka, L., Halonen, M., Moro, A., Kämpe, O., Eskelin, P., Pelto- 909 Huikko, M., and Peltonen, L. (2002) Aire deficient mice develop multiple features of APECED 910 phenotype and show altered immune response. Hum Mol Genet. 11, 397-409. 911 76. Jefferies, C., Wynne, C., and Higgs, R. (2011). Antiviral TRIMs: friend or foe in autoimmune 912 and autoinflammatory disease? Nat Rev Immunol 11, 617-625. 913 77. Heath, W.R., and Carbone, F.R. (2009) Dendritic cell subsets in primary and secondary T cell 914 responses at body surfaces. Nat Immunol. 10,1237-44. 915 78. Bratland, E., Skinningsrud, B., Undlien, D.E., Mozes, E., and Husebye, E.S. (2009)T cell 916 responses to steroid cytochrome P450 21-hydroxylase in patients with autoimmune primary 917 adrenal insufficiency. J Clin Endocrinol Metab.12:5117-24. 918 79. Rottembourg, D., Deal, C., Lambert, M., Mallone, R., Carel, J.C., Lacroix, A., Caillat-Zucman, 919 S., and le Deist, F. (2010) 21-Hydroxylase epitopes are targeted by CD8 T cells in autoimmune 920 Addison's disease. J Autoimmun. 35, 309-315. 921 80. Si, L., Whiteside, T.L., Van Thiel, D.H., Rabin, B.S. (1984). Lymphocyte subpopulations at the 922 site of "piecemeal" necrosis in end stage chronic liver diseases and rejecting liver allografts in 923 cyclosporine-treated patients. Lab Invest 50, 341-347. 924 81. Ryan, K.R., Lawson, C.A., Lorenzi, A.R., Arkwright, P.D., Isaacs, J.D., and Lilic, D. (2005) 925 CD4+CD25+ T-regulatory cells are decreased in patients with autoimmune polyendocrinopathy 926 candidiasis ectodermal dystrophy. J Allergy Clin Immunol 116,1158-1159. 927 82. Saitoh, O., Abiru, N., Nakahara, M., and Nagayama, Y. (2007). CD8+CD122+ T cells, a newly 928 identified regulatory T subset, negatively regulate Graves' hyperthyroidism in a murine model. 929 Endocrinology 148, 6040-6046. 930 83. Kekäläinen, E., Tuovinen, H., Joensuu, J., Gylling, M., Franssila, R., Pöntynen, N., Talvensaari, 931 K., Perheentupa, J., Miettinen, A., and Arstila, T.P. (2007). A defect of regulatory T cells in 932 patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. J Immunol 933 178, 1208-1215. 934 84. Wolff, A.S., Oftedal, B.E., Kisand, K., Ersvaer, E., Lima, K., and Husebye, E.S. (2010) Flow 935 cytometry study of blood cell subtypes reflects autoimmune and inflammatory processes in 936 autoimmune polyendocrine syndrome type I. Scand J Immunol. 71, 459-467. 937 85. Laakso, S.M., Kekäläinen, E., Rossi, L.H., Laurinolli, T.T., Mannerström, H., Heikkilä, N., 938 Lehtoviita A, Perheentupa, J., Jarva, H., and Arstila, T.P. (2011). IL-7 dysregulation and loss of 23

939 CD8+ T cell homeostasis in the monogenic human disease autoimmune polyendocrinopathy- 940 candidiasis-ectodermal dystrophy. J Immunol 187, 2023-2030. 941 86. Laakso SM, Laurinolli TT, Rossi LH, Lehtoviita A, Sairanen H, Perheentupa J, Kekäläinen E, 942 and Arstila, T.P. (2010). Regulatory T cell defect in APECED patients is associated with loss of 943 naive FOXP3(+) precursors and impaired activated population. J Autoimmun 35, 351-357. 944 87. Lehtoviita, A., Rossi, L.H., Kekäläinen, E., Sairanen, H., and Arstila, T.P. (2009). The 945 CD4(+)CD8(+) and CD4(+) subsets of FOXP3(+) thymocytes differ in their response to growth 946 factor deprivation or stimulation. Scand J Immunol 70, 377-383. 947 88. Endharti, A.T., Okuno, Y., Shi, Z., Misawa, N., Toyokuni, S., Ito, M., Isobe, K., and Suzuki, H. 948 (2011) CD8+CD122+ regulatory T cells (Tregs) and CD4+ Tregs cooperatively prevent and 949 cure CD4+ cell-induced colitis. J Immunol. 186:41-52. 950 89. Torgerson, T.R., Linane, A., Moes, N., Anover, S., Mateo, V., Rieux-Laucat, F., Hermine, O., 951 Vijay, S., Gambineri, E., Cerf-Bensussan, N., Fischer, A., Ochs, H.D., Goulet, O., and 952 Ruemmele, F.M. (2007) Severe food allergy as a variant of IPEX syndrome caused by a deletion 953 in a noncoding region of the FOXP3 gene. Gastroenterology. 132, 1705-1717. 954 90. d'Hennezel, E., Ben-Shoshan, M., Ochs, H.D., Torgerson, T.R., Russell, L.J., Lejtenyi, C., 955 Noya, F.J., Jabado, N., Mazer, B., and Piccirillo, C.A. (2009) FOXP3 forkhead domain mutation 956 and regulatory T cells in the IPEX syndrome. N Engl J Med. 361,1710-1713. 957 91. Bacchetta, R., Passerini, L., Gambineri, E., Dai, M., Allan, S.E., Perroni, L., Dagna-Bricarelli, 958 F., Sartirana, C., Matthes-Martin, S., Lawitschka, A., Azzari, C.,Ziegler, S.F., Levings, M.K., 959 and Roncarolo, M.G. (2006) Defective regulatory and effector T cell functions in patients with 960 FOXP3 mutations. J Clin Invest. 116, 1713-1722. 961 92. Hannigan, N.R., Jabs, K., Perez-Atayde, A.R., and Rosen, S. (1996). Autoimmune interstitial 962 nephritis and hepatitis in polyglandular autoimmune syndrome. Pediatr Nephrol 10, 511-514. 963 964 965 966 967 Legend to figure 1. Medullary epithelia cells in thymus, expressing the AIRE proteins 968 (reddish brown), in close vicinity of the Hassall’s corpuscles (HC) where auto-reactive T-cells 969 are thought to be destroyed. Note cell debris in HC. Magnification 1:40. AIRE was 970 demonstrated with specific monoclonal antibody at 1:2000 dilution. Figure 1.JPEG Copyright of Frontiers in Immunology is the property of Frontiers Media S.A. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.